Modified HCV peptide vaccines

ABSTRACT

Provided are an isolated peptide having the amino acid sequence DLMGYIPAV (SEQ ID NO: 1), an isolated HCV core polypeptide comprising an L→A substitution at amino acid position 139, an isolated HCV core polypeptide having the amino acid sequence of SEQ ID NO: 2, and a fragment of an HCV core polypeptide having fewer amino acids than the entire HCV core polypeptide and comprising the amino acid sequence of SEQ ID NO:1. Also provided are nucleic acids which encode the peptides and polypeptides of this invention, vectors comprising the nucleic acids of this invention and cells comprising the vectors and nucleic acids of this invention. Further provided are methods of producing an immune response in a subject and/or treating or preventing HCV infection in a subject, comprising administering to the subject, or to a cell of the subject, any of the compositions of this invention.

This application is a divisional application of and claims benefit ofU.S. application Ser. No. 09/763,260, filed Oct. 19, 2001, now U.S. Pat.No. 6,685,944, which was the national stage of International ApplicationPCT/US99/18674, filed Aug. 17, 1999, which claims benefit of U.S.Provisional Application No. 60/097,446, filed Aug. 21, 1998, both ofwhich are incorporated herein in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of viral vaccines. Inparticular, the present invention relates to modification of immunogenicepitopes of the hepatitis C virus (HCV) core protein to elicit anenhanced immune response against HCV.

2. Background Art

Hepatitis C virus (HCV) is a single stranded RNA virus responsible forthe majority of non-A non-B hepatitis (1). Infection by HCV frequentlyevolves to chronicity and in many cases leads to liver cirrhosis andhepatocellular carcinoma (2). The cellular immune response is thought tobe responsible for viral clearance in many viral infections (3–7) and inthe case of HCV, a cytotoxic T lymphocyte (CTL) response is present inacutely and chronically infected patients (8–14), but its role in viralclearance has not been elucidated. CTL responses have been detected inperipheral blood mononuclear cells (PBMC) and in intrahepaticlymphocytic infiltrate in patients with chronic hepatitis (15) and inthe liver of infected chimpanzees (16), indicating that in these casesthe virus is able to persist despite this immune response (17). Thereasons for the inadequacy of this immune response in chronicallyinfected patients are not known (18).

CD8⁺ CTL recognize antigens as peptides presented by class I moleculesof the major histocompatibility complex (MHC) on the cell surface. Thesepeptides are usually 8–10 amino acids long and are generated afterprocessing of intracellular antigens (3,19–21). Analysis of peptidespresented by MHC class I molecules has led to the definition of severalsequence patterns or motifs (22–24) for peptides that bind to eachparticular MHC allele or group of alleles (supermotif) (25). Thesemotifs are based on the presence in precise positions in the peptidesequence of several amino acids (agretopic residues) called anchorresidues (22,26), responsible for interactions between peptide and MHCmolecule, as well as other secondary positions that may help instabilizing these interactions (27–29). The use of these motifs toidentify peptides able to bind to MHC molecules, together with thedevelopment of MHC-peptide binding assays, has led to thecharacterization of many CTL epitopes in the HCV polypeptide presentedby different MHC molecules (9,10,12,30). Among the best studied motifsis that of HLA-A2.1, which is prevalent in a high percentage of thepopulation (31). Several reports describe the binding motif for thisallele, pointing out the importance of anchor as well as secondaryresidues. Also, MHC binding has been correlated with immunogenicity indifferent mouse and human systems (30,32–37).

Despite the presence of the typical anchor residues, the bindingcapability of a peptide epitope may vary, depending on the othersecondary residues. Thus, the presence of certain amino acids insecondary positions may enhance or impair a peptide=s binding ability(28,38,39). Other amino acids (epitopic residues) are responsible forrecognition by the T cell receptor (TCR). Thus, T cell response istriggered by interactions in the trimolecular complex: MHC-antigenicpeptide-TCR, together with other co-stimulatory molecules (40,41). Theseinteractions occur between the antigenic peptide and pockets in thestructure of both MHC and TCR molecules and changes in the amino acidsequence of the peptide may affect any of these interfaces.

Because of the inadequacy of the immune response in HCV-infectedindividuals, there exists a great need to enhance the immune response toHCV immunogenic epitopes without impairing MHC binding affinity or Tcell recognition. The present invention overcomes the previouslimitations and shortcomings in the art by providing immunogenicpeptides of HCV core protein which elicit an enhanced immune response,methods for making these peptides and methods for using these peptidesfor a variety of therapeutic, diagnostic and prognostic applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. Binding of C7A2 Alanine (A) and non-Alanine(B)-substituted peptides to TAP-deficient HLA-A2.1. T2 cells wereincubated overnight in a 96 well plate in CTM with 10 Φg/ml humanβ2-microglobulin and different peptide concentrations. The next day,cells were washed and HLA-A2.1 expression was assessed by flow cytometryusing the anti HLA-A2 BB7.2 monoclonal antibody and FITC labeled goatanti mouse Ig. Results are expressed as fluorescence index (FI),calculated according to the formula: (mean fluorescence withpeptide−mean fluorescence without peptide)/mean fluorescence withoutpeptide. Background fluorescence without BB7.2 was subtracted for eachindividual value. FI_(0.5) represents those peptide concentrations thatincrease HLA-A2.1 expression by 50% over the no peptide control and werecalculated from the titration curves for each particular peptide.

FIGS. 2A and 2B. Recognition of C7A2 Alanine (A) and non-Alanine(B)-substituted peptides by human CTL line ViT2, which is specific forC7A2 peptide presented by HLA-A2.1. C1R.A2.1 target cells were incubatedfor 2 h with ⁵¹Cr, washed three times and plated at 3000 cells per wellin 96 well round bottom plates with different peptide concentrations.After 2 h, effector cells were added (E/T ratio: 5/1) and the plateswere incubated for 4 h.

FIGS. 3A–D. Recognition of C7A2 Alanine (A,C) and non-Alanine(B,D)-substituted peptides by transgenic AAD mouse CTL lines AAD. 10(A,B) and AAD. 1 (C,D) after 10–12 in vitro stimulations. C1R.AAD targetcells were incubated for 2 h with ⁵¹Cr, washed three times and plated at3000 cells per well in 96 well round bottom plates with differentpeptide concentrations. After 2 h, effector cells were added (E/T ratio:10/1) and the plates were incubated for 4 h.

FIG. 4. Immunogenicity of C7A2-substituted peptides in AAD transgenicmice. Mice were immunized with 50 nmol CTL epitope plus 50 nmol HBVc128–140 helper epitope and GM-CSF and IL-12 in IFA. Two weeks later,mice were boosted and 10–14 days after the boost, spleen cells wereremoved and stimulated in vitro with the CTL epitope. After one week ofin vitro stimulation, a CTL assay was performed with target cells (AADCon A blasts) incubated with or without 10 ΦM CTL peptide. CTL from eachgroup were tested only with the peptide used to immunize those mice.Only results for peptide-pulsed target cells are shown. In all cases,percentage of specific lysis against peptide-unpulsed target cells wasbelow 5%.

FIGS. 5A and 5B. Induction of CTL immune response against C7A2-WT in AADtransgenic mice using different C7A2 peptide variants. Mice wereimmunized with 50 or 15 mmol CTL epitope C7A2-WT (A) or 8A (B) plus 50mmol HBVc 128–140 helper epitope and GM-CSF and IL-12 in IFA. Two weekslater, mice were boosted under the same conditions and 10–14 days afterthe boost, spleen cells were removed and stimulated in vitro with 10 ΦMCTL peptide-pulsed spleen cells. After two weeks of in vitrostimulation, a CTL assay was performed with target cells (AAD Con Ablasts) incubated with or without 10 ΦM peptide. The numbers 50.1 and50.2, and 15.1 and 15.2 designate different mice immunized with 50 or 15nmol CTL epitope respectively. (E/T ratio in the assay: 90/1).

FIG. 6. Induction of CTL immune response against C7A2-WT in A2 Kbtransgenic mice using different C7A2 peptide variants. Mice wereimmunized with 50 nmol CTL epitope C7A2-WT or 8A plus 50 nmol HBVc128–140 helper epitope in IFA. Two weeks later, mice were boosted underthe same conditions and 10–14 days after the boost, spleen cells wereremoved and stimulated in vitro with 10 ΦM CTL peptide-pulsed spleencells. After two weeks of in vitro stimulation, a CTL assay wasperformed with target cells (A2 Kb Con A blasts) incubated with orwithout 10 ΦM peptide. WT.1 and WT.2, and 8A.1 and 8A.2 designatedifferent mice immunized with C7A2-WT and 8A CTL epitope respectively.(E/T ratio in the assay: 120/1 for WT. 1 and WT.2; 70/1 for 8A.1 and8A.2).

FIG. 7. Avidity of short term CTL lines induced with C7A2-WT andC7A2-8A. AAD transgenic mice were immunized as described herein with 50nmol CTL epitope C7A2-WT (solid lines) or C7A2-8A (dotted lines) plus 50nmol HBVc 128–140 helper epitope and GM-CSF and IL-12 in IFA. Two weekslater, mice were boosted under the same conditions and 10–14 days afterthe boost, spleen cells were removed and stimulated in vitro with 10 ΦMCTL peptide-pulsed spleen cells. After 3–4 in vitro stimulation cycles,a CTL assay was performed with target cells (C1R.AAD) incubated withdifferent peptide concentrations as indicated (circles, targets withC7A2-WT peptide; squares, targets with C7A2-8A peptide), at an E: Tratio of 10:1.

FIGS. 8A and 8B. Recognition of C7A2-derived peptides from different HCVgenotypes by CTL induced following immunization with peptide 8A. AAD (A)and A2 Kb (B) transgenic mice were immunized with 50 nmol CTL epitope 8Aplus 50 nmol HBVc 128–140 helper epitope in IFA. Two weeks later, micewere boosted under the same conditions and 10–14 days after the boost,spleen cells were removed and stimulated in vitro with 10 ΦM CTLpeptide-pulsed spleen cells. After two weeks of in vitro stimulation, aCTL assay was performed with target cells (Con A blasts) incubated withthe different peptides at 10 ΦM. C7A2-WT has a sequence from HCVgenotypes 1a, 1b and 3a, whereas peptide C7A2-8V has a sequence fromgenotypes 2a and 2b.

FIG. 9. Comparison of CTL activity at four weeks between AC7 30 μg andAC7-8A 30 μg immunization to AAD mice. Target (T)=3000.

SUMMARY OF THE INVENTION

The present invention provides 1) an isolated peptide having the aminoacid sequence DLMGYIPAV, (SEQ ID NO: 1); 2) an isolated HCV corepolypeptide comprising an L6A substitution at amino acid position 139;3) an isolated HCV core polypeptide having the amino acid sequence ofSEQ ID NO: 2; and 4) a fragment of an HCV core polypeptide having feweramino acids than the entire HCV core polypeptide and comprising theamino acid sequence of SEQ ID NO:1. Also provided are nucleic acidswhich encode the peptides and polypeptides of this invention and vectorscomprising the nucleic acids of this invention. These compositions canbe present in a pharmaceutically acceptable carrier, which can alsoinclude a suitable adjuvant.

In addition, the present invention provides a method of producing an HCVcore peptide having enhanced immunogenicity, comprising substituting oneor more amino acids of the peptide having the amino acid sequenceDLMGYIPLV (SEQ ID NO:3) and detecting enhanced immunogenicity of thesubstituted peptide as compared to the immunogenicity of a controlpeptide having the amino acid sequence of DLMGYIPLV (SEQ ID NO:3),whereby a substituted peptide having greater immunogenicity than thecontrol peptide is a HCV core peptide having enhanced immunogenicity.

The present invention further provides methods of producing an immuneresponse in a subject and treating or preventing HCV infection in asubject comprising administering to the subject, or to a cell of thesubject, any of the compositions of this invention.

The present invention also provides a method of activating cytotoxic Tlymphocytes, comprising contacting the lymphocytes with any of thepeptides or polypeptides of this invention in the presence of a class Imajor histocompatibility complex molecule and also provides acomposition comprising cytotoxic T lymphocytes activated by contact withthe peptides or polypeptides of this invention in the presence of aclass I major histocompatibility complex molecule.

Also provided are methods of detecting the presence of HCV corepolypeptide in a cell comprising contacting the cell with the activatedcytotoxic T lymphocytes of this invention under conditions wherebycytolysis of target cells can occur or cytokine production of thelymphocytes can occur and detecting cytolysis of target cells orcytokine production of the lymphocytes, whereby the detection ofcytolysis of target cells or cytokine production of the lymphocytesindicates the presence of HCV core polypeptide in the cell.

In addition, the present invention provides a method of detectinghepatitis C virus in a sample comprising: a) contacting the sample witha cell which is susceptible to infection by hepatitis C virus underconditions whereby the cell can be infected by hepatitis C virus in thesample; D) contacting the cell of step (a) with the cytolytic Tlymphocytes of this invention under conditions whereby cytolysis oftarget cells or cytokine production of the lymphocytes can occur; and c)detecting cytolysis of target cells or cytokine production of thelymphocytes, whereby the detection of cytolysis of target cells orcytokine production of the lymphocytes indicates the presence ofhepatitis C virus in the sample.

The present invention further provides a method of diagnosing HCVinfection in a subject comprising contacting cytotoxic T lymphocytes ofthe subject with the peptides or polypeptides of this invention in thepresence of a class I major histocompatibility complex molecule underconditions whereby cytolysis of target cells or cytokine production ofthe lymphocytes can occur and detecting cytolysis of target cells orcytokine production of the lymphocytes, whereby the detection ofcytolysis of target cells or cytokine production of the lymphocytesindicates a diagnosis of HCV infection in the subject.

Further provided are methods of determining a viral load of HCV in asubject comprising a) serially diluting a biological sample from thesubject which contains hepatitis C virus; b) contacting each seriallydiluted sample with a cell which is susceptible to infection byhepatitis C virus under conditions whereby the cell can be infected byhepatitis C virus in the sample; c) contacting the cell of step (b) withthe activated cytolytic T lymphocytes of this invention under conditionswhereby cytolysis of target cells or cytokine production in thelymphocytes can occur; measuring the amount of cytolysis of target cellsor cytokine production in the lymphocytes; comparing the amount ofcytolysis of target cells or cytokine production in the lymphocytes withthe amount of cytolysis of target cells or cytokine production byactivated cytotoxic T lymphocytes contacted with cells infected withserially diluted control samples containing a known amount of hepatitisC virus; and determining the viral load of hepatitis C virus in thesubject from the comparison with the samples of known amount.

Finally, the present invention provides a method of determining theprognosis of a subject diagnosed with hepatitis C virus infection,comprising determining a viral load for the subject according to themethods of this invention, whereby a high viral load indicates a poorprognosis and a low viral load indicates a good prognosis.

Various other objectives and advantages of the present invention willbecome apparent from the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “a” or “an” may mean one or more. For example, “a” cellmay mean one cell or more than one cell.

The present invention is based on the unexpected and surprisingdiscovery, through the process of epitope enhancement, of an HCV corepeptide having the amino acid sequence DLMGYIPAV (SEQ ID NO:1), that hasenhanced HLA-A2 binding affinity and CTL recognition in vitro andenhanced immunogenicity in vivo when compared to the naturally occurringpeptide DLMGYIPLV (SEQ ID NO:3) at positions 132–140 in the HCV corepolypeptide. Thus, the present invention provides an isolated peptidehaving the amino acid sequence of SEQ ID NO:1.

The present invention also provides an isolated HCV core polypeptidecomprising an L6A substitution at amino acid position 139. Thus, the HCVcore polypeptide of this invention can have the amino acid sequence ofany of the variants of HCV now known or identified in the future andfurther comprise an L6A substitution at amino acid position 139 (see,e.g., ref. 87). Furthermore, the present invention provides an isolatedHCV core polypeptide having the amino acid sequence of SEQ ID NO: 2.

Further, the present invention provides a polypeptide fragment of theHCV core polypeptide, having fewer amino acids than the HCV corepolypeptide and comprising the amino acid sequence of SEQ ID NO: 1.Thus, the polypeptide fragment of this invention can comprise asufficient number of contiguous amino acids to be identified as apolypeptide fragment of the HCV core polypeptide and including the aminoacid sequence of SEQ ID NO:1, yet does not contain all of the aminoacids of the HCV core polypeptide. For example, the entire HCV corepolypeptide (SEQ ID NO:4) has 191 amino acids. Thus, the polypeptidefragment of this invention can have any number of amino acids fewer than191 amino acids (e.g., 191-1, 191-5, 191-10, 191-50, 191-100, 191-130,191-180 etc.) and having at least three amino acids which are contiguousamino acids of the HCV core polypeptide and would be recognized by oneof ordinary skill in the art as contiguous amino acids of the HCV corepolypeptide according to standard methods for identifying polypeptidesand would also include the amino acid sequence of SEQ ID NO:1. The aminoacid sequence of SEQ ID NO:1 can be present in the polypeptide fragmentof this invention once or multiple times and can be at any position(e.g., N terminal, C terminal, internal) relative to the other aminoacid sequence of the fragment.

“Isolated” as used herein means the peptide or polypeptide of thisinvention is sufficiently free of contaminants or cell components withwhich peptides or polypeptides normally occur and is present in suchconcentration as to be the only significant peptide or polypeptidepresent in the sample. “Isolated” does not mean that the preparation istechnically pure (homogeneous), but it is sufficiently pure to providethe peptide or polypeptide in a form in which it can be usedtherapeutically.

“Epitope” as used herein means a specific amino acid sequence of limitedlength which, when present in the proper conformation, provides areactive site for an antibody or T cell receptor. The identification ofepitopes on antigens can be carried out by immunology protocols that arestandard in the art (79).

Also as used herein, the terms peptide and polypeptide are used todescribe a chain of amino acids which correspond to those encoded by anucleic acid. A peptide usually describes a chain of amino acids of fromtwo to about 30 amino acids and polypeptide usually describes a chain ofamino acids having more than about 30 amino acids. The term polypeptidecan refer to a linear chain of amino acids or it can refer to a chain ofamino acids which have been processed and folded into a functionalprotein. It is understood, however, that 30 is an arbitrary number withregard to distinguishing peptides and polypeptides and the terms may beused interchangeably for a chain of amino acids around 30. The peptidesand polypeptides of the present invention are obtained by isolation andpurification of the peptides and polypeptides from cells where they areproduced naturally or by expression of exogenous nucleic acid encodingthe peptide or polypeptide. The peptides and polypeptides of thisinvention can be obtained by chemical synthesis, by proteolytic cleavageof a polypeptide and or by synthesis from nucleic acid encoding thepeptide or polypeptide.

The main discovery of this invention is a peptide or polypeptide havingamino acid substitutions which enhance immunogenicity. Suchsubstitutions can be made in the peptides and polypeptides of thisinvention by methods standard in the art and as set forth herein andenhanced immunogenicity can be determined according to the methodsprovided in the Examples herein. It is also understood that the peptidesand polypeptides of this invention may also contain conservativesubstitutions where a naturally occurring amino acid is replaced by onehaving similar properties and which does not alter the function of thepolypeptide. Such conservative substitutions are well known in the art.Thus, it is understood that, where desired, modifications and changes,which are distinct from the substitutions which enhance immunogenicity,may be made in the nucleic acid and/or amino acid sequence of thepeptides and polypeptides of the present invention and still obtain apeptide or polypeptide having like or otherwise desirablecharacteristics. Such changes may occur in natural isolates or may besynthetically introduced using site-specific mutagenesis, the proceduresfor which, such as mis-match polymerase chain reaction (PCR), are wellknown in the art.

Moreover, the present invention provides isolated nucleic acids encodingeach of the following: 1) the peptide having the amino acid sequence ofSEQ ID NO:1; 2) the HCV core polypeptide having the amino acid sequenceof SEQ ID NO:2; 3) the HCV core polypeptide comprising an L6Asubstitution at amino acid position 139; and 4) a fragment of the HCVcore polypeptide having fewer amino acids than the entire HCV corepolypeptide and comprising the peptide having the amino acid sequence ofSEQ ID NO:1.

“Nucleic acid” as used herein refers to single- or double-strandedmolecules which may be DNA, comprised of the nucleotide bases A, T, Cand G, or RNA, comprised of the bases A, U (substitutes for T), C, andG. The nucleic acid may represent a coding strand or its complement.Nucleic acids may be identical in sequence to the sequence which isnaturally occurring or may include alternative codons which encode thesame amino acid as that which is found in the naturally occurringsequence. Furthermore, nucleic acids may include codons which representconservative substitutions of amino acids as are well known in the art.

As used herein, the term “isolated nucleic acid” means a nucleic acidseparated or substantially free from at least some of the othercomponents of the naturally occurring organism, for example, the cellstructural components commonly found associated with nucleic acids in acellular environment and/or other nucleic acids. The isolation ofnucleic acids can therefore be accomplished by techniques such as celllysis followed by phenol plus chloroform extraction, followed by ethanolprecipitation of the nucleic acids (80). The nucleic acids of thisinvention can be isolated from cells according to methods well known inthe art for isolating nucleic acids. Alternatively, the nucleic acids ofthe present invention can be synthesized according to standard protocolswell described in the literature for synthesizing nucleic acids.Modifications to the nucleic acids of the invention are alsocontemplated, provided that the essential structure and function of thepeptide or polypeptide encoded by the nucleic acid are maintained.

The nucleic acid encoding the peptide or polypeptide of this inventioncan be part of a recombinant nucleic acid construct comprising anycombination of restriction sites and/or functional elements as are wellknown in the art which facilitate molecular cloning and otherrecombinant DNA manipulations. Thus, the present invention furtherprovides a recombinant nucleic acid construct comprising a nucleic acidencoding a peptide and/or polypeptide of this invention.

The present invention further provides a vector comprising a nucleicacid encoding a peptide and/or polypeptide of this invention. The vectorcan be an expression vector which contains all of the genetic componentsrequired for expression of the nucleic acid in cells into which thevector has been introduced, as are well known in the art. The expressionvector can be a commercial expression vector or it can be constructed inthe laboratory according to standard molecular biology protocols. Theexpression vector can comprise viral nucleic acid including, but notlimited to, vaccinia virus, adenovirus, retrovirus and/oradeno-associated virus nucleic acid. The nucleic acid or vector of thisinvention can also be in a liposome or a delivery vehicle which can betaken up by a cell via receptor-mediated or other type of endocytosis.

The nucleic acid of this invention can be in a cell, which can be a cellexpressing the nucleic acid whereby a peptide and/or polypeptide of thisinvention is produced in the cell. In addition, the vector of thisinvention can be in a cell, which can be a cell expressing the nucleicacid of the vector whereby a peptide and/or polypeptide of thisinvention is produced in the cell. It is also contemplated that thenucleic acids and/or vectors of this invention can be present in a hostanimal (e.g., a transgenic animal) which expresses the nucleic acids ofthis invention and produces the peptides and/or polypeptides of thisinvention.

The nucleic acid encoding the peptides and polypeptides of thisinvention can be any nucleic acid that functionally encodes the peptidesand polypeptides of this invention. To functionally encode the peptidesand polypeptides (i.e., allow the nucleic acids to be expressed), thenucleic acid of this invention can include, for example, expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer and necessary information processing sites, such as ribosomebinding sites, RNA splice sites, polyadenylation sites andtranscriptional terminator sequences.

Preferred expression control sequences are promoters derived frommetallothionine genes, actin genes, immunoglobulin genes, CMV, SV40,adenovirus, bovine papilloma virus, etc. A nucleic acid encoding aselected peptide or polypeptide can readily be determined based upon thegenetic code for the amino acid sequence of the selected peptide orpolypeptide and many nucleic acids will encode any selected peptide orpolypeptide. Modifications in the nucleic acid sequence encoding thepeptide or polypeptide are also contemplated. Modifications that can beuseful are modifications to the sequences controlling expression of thepeptide or polypeptide to make production of the peptide or polypeptideinducible or repressible as controlled by the appropriate inducer orrepressor. Such methods are standard in the art (81). The nucleic acidof this invention can be generated by means standard in the art, such asby recombinant nucleic acid techniques and by synthetic nucleic acidsynthesis or in vitro enzymatic synthesis.

The present invention further provides a method of producing an HCV corepeptide having enhanced immunogenicity, comprising substituting one ormore amino acids of the peptide having amino acid sequence DLMGYIPLV(SEQ ID NO:3) and detecting enhanced immunogenicity of the substitutedpeptide as compared to the immunogenicity of a control peptide havingthe amino acid sequence of DLMGYIPLV (SEQ ID NO:3), whereby asubstituted peptide having greater immunogenicity than the controlpeptide is an HCV core peptide having enhanced immunogenicity.Immunogenicity of the substituted and control peptide is determinedaccording to the methods set forth in the Examples provided herein. Theproduction of the peptide having the amino acid sequence of SEQ ID NO:3and the substituted peptides of this invention can be carried outaccording to methods standard in the art for peptide synthesis.Alternatively, a nucleic acid encoding the amino acid sequence of SEQ IDNO:3 or encoding a substituted peptide of interest can be synthesizedaccording to standard nucleic acid synthesis protocols and the nucleicacid can be expressed according to methods well known for expression ofnucleic acid. The resulting peptide can then be removed from theexpression system by standard isolation and purification procedures andtested for immunogenicity as taught herein.

On the basis of the discovery of this invention, substitution of anamino acid or amino acids of the peptide DLMGYIPLV (SEQ ID NO:3) toproduce a peptide of this invention having enhanced immunogenicity asdetermined by the methods of this invention, can be carried out by asystematic approach comprising replacement of each of the amino acids inthe peptide sequentially, starting from the amino terminus of thepeptide. Peptides having a single amino acid substitution which showenhanced immunogenicity by the methods described herein can beidentified and a second amino acid substitution can be introduced intothe amino acid sequence of the singly-substituted peptide sequentially,starting from the amino terminus of the singly-substituted peptide.These doubly-substituted peptides can then be tested for enhancedimmunogenicity and those which show such enhancement can have furtheramino acid substitutions made by the systematic, sequential methoddescribed herein. Thus, any combination of substitutions can be testedfor enhanced immunogenicity in a systematic manner.

The present invention also provides a method for producing the peptidesand polypeptides of this invention comprising producing the cells ofthis invention which contain the nucleic acids or vectors of thisinvention as exogenous nucleic acid; culturing the cells underconditions whereby the exogenous nucleic acid in the cell can beexpressed and the encoded peptide and/or polypeptide can be produced;and isolating the peptide and/or polypeptide from the cell. Thus, it iscontemplated that the peptides and polypeptides of this invention can beproduced in quantity in vitro in either prokaryotic or eukaryoticexpression systems as are well known in the art.

For expression in a prokaryotic system, there are numerous E. coli(Escherichia coli) expression vectors known to one of ordinary skill inthe art useful for the expression of nucleic acid which encodes peptidesor polypeptides. Other microbial hosts suitable for use include bacilli,such as Bacillus subtilis, and other enterobacteria, such as Salmonella,Serratia, as well as various Pseudomonas species. These prokaryotichosts can support expression vectors which will typically containexpression control sequences compatible with the host cell (e.g., anorigin of replication). In addition, any number of a variety ofwell-known promoters will be present, such as the lactose promotersystem, a tryptophan (Trp) promoter system, a beta-lactamase promotersystem, or a promoter system from phage lambda. The promoters willtypically control expression, optionally with an operator sequence andhave ribosome binding site sequences for example, for initiating andcompleting transcription and translation. If necessary, an aminoterminal methionine can be provided by insertion of a Met codon 5′ andin-frame with the polypeptide. Also, the carboxy-terminal extension ofthe polypeptide can be removed using standard oligonucleotidemutagenesis procedures.

The nucleic acid sequences can be expressed in hosts after the sequenceshave been positioned to ensure the functioning of an expression controlsequence. These expression vectors are typically replicable in the hostorganisms either as episomes or as an integral part of the hostchromosomal DNA. Commonly, expression vectors can contain selectionmarkers, e.g., tetracycline resistance or hygromycin resistance, topermit detection and/or selection of those cells transformed with thedesired nucleic acid sequences (82).

For eukaryotic system expression, a yeast expression system can be used.There are several advantages to yeast expression systems. First,evidence exists that polypeptides produced in a yeast expression systemexhibit correct disulfide pairing. Second, post-translationalglycosylation is efficiently carried out by yeast expression systems.The Saccharomyces cerevisiae pre-pro-alpha-factor leader region (encodedby the MFα-1 gene) is routinely used to direct protein secretion fromyeast (83). The leader region of pre-pro-alpha-factor contains a signalpeptide and a pro-segment which includes a recognition sequence for ayeast protease encoded by the KEX2 gene. This enzyme cleaves theprecursor protein on the carboxyl side of a Lys-Arg dipeptidecleavage-signal sequence. The polypeptide coding sequence can be fusedin-frame to the pre-pro-alpha-factor leader region. This construct isthen put under the control of a strong transcription promoter, such asthe alcohol dehydrogenase I promoter or a glycolytic promoter. Thecoding sequence is followed by a translation termination codon, which isfollowed by transcription termination signals. Alternatively, the codingsequence of interest can be fused to a second polypeptide codingsequence, such as Sj26 or β-galactosidase, used to facilitatepurification of the resulting fusion polypeptide by affinitychromatography. The insertion of protease cleavage sites to separate thecomponents of the fusion polypeptide is applicable to constructs usedfor expression in yeast.

Efficient post-translational glycosylation and expression of recombinantpolypeptides can also be achieved in Baculovirus systems in insectcells, as are well known in the art.

The peptides and polypeptides of this invention can also be expressed inmammalian cells. Mammalian cells permit the expression of peptides andpolypeptides in an environment that favors important post-translationalmodifications such as folding and cysteine pairing, addition of complexcarbohydrate structures and secretion of active protein. Vectors usefulfor the expression of peptides and polypeptides in mammalian cells arecharacterized by insertion of the coding sequence between a strong viralpromoter and a polyadenylation signal. The vectors can contain genesconferring either gentamicin or methotrexate resistance for use asselectable markers. For example, the coding sequence can be introducedinto a Chinese hamster ovary (CHO) cell line using a methotrexateresistance-encoding vector. Presence of the vector RNA in transformedcells can be confirmed by Northern blot analysis and production of acDNA or opposite strand RNA corresponding to the peptide or polypeptidecoding sequence can be confirmed by Southern and Northern blot analysis,respectively. A number of other suitable host cell lines capable ofproducing exogenous peptides and polypeptides have been developed in theart and include the CHO cell lines, HeLa cells, myeloma cell lines,Jurkat cells and the like. Expression vectors for these cells caninclude expression control sequences, as described above.

The nucleic acids and/or vectors of this invention can be transferredinto the host cell by well-known methods, which vary depending on thetype of cell host. For example, calcium chloride transfection iscommonly utilized for prokaryotic cells, whereas calcium phosphatetreatment or electroporation may be used for other cell hosts.

The peptides, polypeptides, nucleic acids, vectors and cells of thisinvention can be present in a pharmaceutically acceptable carrier. Thus,the present invention provides a composition comprising 1) a peptidehaving the amino acid sequence of SEQ ID NO: 1; 2) an HCV corepolypeptide comprising an L6A substitution at amino acid position 139;3) an HCV core polypeptide having the amino acid sequence of SEQ IDNO:2; 4) a fragment of an HCV core polypeptide having fewer amino acidsthan the entire HCV core polypeptide and comprising the amino acidsequence of SEQ ID NO:1; 5) a nucleic acid encoding any of the peptidesor polypeptides of this invention; 6) a vector comprising any of thenucleic acids of this invention; and/or 7) a cell comprising any of thenucleic acids and/or vectors of this invention, in a pharmaceuticallyacceptable carrier. By “pharmaceutically acceptable” is meant a materialthat is not biologically or otherwise undesirable, i.e., the materialmay be administered to an individual along with the selected peptide,polypeptide, nucleic acid, vector or cell without causing substantialdeleterious biological effects or interacting in a deleterious mannerwith any of the other components of the composition in which it iscontained.

Furthermore, any of the compositions of this invention can comprise apharmaceutically acceptable carrier and a suitable adjuvant. As usedherein, “suitable adjuvant” describes an adjuvant capable of beingcombined with the peptide or polypeptide of this invention to furtherenhance an immune response without deleterious effect on the subject orthe cell of the subject. A suitable adjuvant can be, but is not limitedto, MONTANIDE ISA51 (Seppic, Inc., Fairfield, N.J.), SYNTEX adjuvantformulation 1 (SAF-1), composed of 5 percent (wt/vol) squalene (DASF,Parsippany, N.J.), 2.5 percent Pluronic, L121 polymer (Aldrich Chemical,Milwaukee), and 0.2 percent polysorbate (Tween 80, Sigma) inphosphate-buffered saline. Other suitable adjuvants are well known inthe art and include QS-21, Freund's adjuvant (complete and incomplete),alum, aluminum phosphate, aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE) and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trealosedimycolate and cell wall skeleton (MPL+TDM+CWS) in 2% squalene/Tween 80emulsion.

The compositions of the present invention can also include othermedicinal agents, pharmaceutical agents, carriers, diluents,immunostimulatory cytokines, etc. Actual methods of preparing suchdosage forms are known, or will be apparent, to those skilled in thisart (84).

It is contemplated that the above-described compositions of thisinvention can be administered to a subject or to a cell of a subject toimpart a therapeutic benefit. Thus, the present invention furtherprovides a method of producing an immune response in an immune cell of asubject, comprising contacting the cell with 1) a peptide having theamino acid sequence of SEQ ID NO:1; 2) an HCV core polypeptide havingthe amino acid sequence of SEQ ID NO:2; 3) an HCV core polypeptidecomprising an L6A substitution at amino acid position 139; 4) a fragmentof the HCV core polypeptide having fewer amino acids than a complete HCVcore polypeptide and comprising SEQ ID NO:1; 5) a nucleic acid encodingany of the peptides or polypeptides of this invention and/or 6) a vectorcomprising any one of the nucleic acids of this invention. The cell canbe in vivo or ex vivo and can be, but is not limited to a CD8+ Tlymphocyte (e.g., a cytotoxic T lymphocyte) or an MHC I-expressingantigen presenting cell, such as a dendritic cell, a macrophage or amonocyte.

The present invention additionally provides a method of producing animmune response in a subject by administering to the subject any of thecompositions of this invention, including a composition comprising apharmaceutically acceptable carrier and 1) a peptide having the aminoacid sequence of SEQ ID NO:1; 2) an HCV core polypeptide having theamino acid sequence of SEQ ID NO:2; 3) an HCV core polypeptidecomprising an L6A substitution at amino acid position 139; 4) a fragmentof the HCV core polypeptide having fewer amino acids than a complete HCVcore polypeptide and comprising SEQ ID NO:1; 5) a nucleic acid encodingany of the peptides or polypeptides of this invention and/or 6) a vectorcomprising any one of the nucleic acids of this invention. Thecomposition can further comprise a suitable adjuvant, as set forthherein.

Detection of an immune response in the subject or in the cells of thesubject can be carried out according to the methods set forth in theExamples provided herein, such as for detecting the presence ofcytotoxic T cells activated by the peptides or polypeptides of thisinvention.

In addition, the present invention provides a method of treating orpreventing HCV infection in a subject comprising contacting an immunecell of the subject with any of the peptide, polypeptide, fragments,nucleic acids vectors and/or cells of this invention. The cell can be invivo or ex vivo and can be a CD8⁺ T cell which is contacted with thepeptide or polypeptide of this invention in the presence of a class IMHC molecule, which can be a soluble molecule or it can be present onthe surface of a cell which expresses class I MHC molecules. The cellcan also be an antigen presenting cell or other class I MHC-expressingcell which can be contacted with the nucleic acids and/or vectors ofthis invention under conditions whereby the nucleic acid or vector isintroduced into the cell by standard methods for uptake of nucleic acidand vectors. The nucleic acid encoding the peptide or polypeptide ofthis invention is then expressed and the peptide or polypeptide productis processed within the antigen presenting cell or other MHCI-expressing cell and presented on the cell surface as an MHC I/antigencomplex. The antigen presenting cell or other class I MHC-expressingcell is then contacted with an immune cell of the subject which bindsthe class I MHC/antigen complex and elicits an immune response whichtreats or prevents HCV infection in the subject.

The present invention also provides a method of treating or preventingHCV infection in a subject, comprising administering to the subject anyof the compositions of this invention, including a compositioncomprising a pharmaceutically acceptable carrier and 1) a peptide havingthe amino acid sequence of SEQ ID NO:1; 2) an HCV core polypeptidehaving the amino acid sequence of SEQ ID NO:2; 3) an HCV corepolypeptide comprising an L6A substitution at amino acid position 139;4) a fragment of the HCV core polypeptide having fewer amino acids thana complete HCV core polypeptide and comprising SEQ ID NO:1; 5) a nucleicacid encoding any of the peptides or polypeptides of this inventionand/or 6) a vector comprising any one of the nucleic acids of thisinvention.

As set forth above, it is contemplated that in the methods wherein thecompositions of this invention are administered to a subject or to acell of a subject, such methods can further comprise the step ofadministering a suitable adjuvant to the subject or to a cell of thesubject. The adjuvant can be in the composition of this invention or theadjuvant can be in a separate composition comprising the suitableadjuvant and a pharmaceutically acceptable carrier. The adjuvant can beadministered prior to, simultaneous with or after administration of thecomposition containing any of the peptides, polypeptides, nucleic acidsand/or vectors of this invention. For example, QS-21, similar to alum,complete Freund=s adjuvant, SAF, etc., can be administered within hours(before or after) of administration of the peptide. The effectiveness ofan adjuvant can be determined by measuring the immune response directedagainst the peptide or polypeptide of this invention with and withoutthe adjuvant, using standard procedures, as described in the Examplesherein.

The subject of this invention can be any subject in need of the immuneresponse of this invention and/or in need of treatment for or preventionfrom HCV infection. Symptoms of HCV infection can include low-gradefever, malaise and anorexia. Elevation of serum liver enzymes such asSGOT and SGPT is also typically found. The diagnosis of HCV infectioncan be made by detecting HCV antibodies in the subject=s serum.Confirmation of the diagnosis can be made by PCR assay of the subject=sblood for HCV RNA. It would also be well understood in the art that manysubjects infected with HCV are asymptomatic.

Common sources of infection can include blood transfusions, infectedsexual partners and contaminated needles. Further, subjects vulnerableto blood-borne disease can be at risk for HCV infection. Thus, a subjectfor whom the methods of this invention would be indicated for preventingHCV infection can be a subject who has received a blood transfusion, hasan infected sexual partner, shares contaminated needles for intravenousdrug abuse or has been accidentally stuck with a contaminated needle orexposed to HCV-contaminated tissues or body fluids (e.g., a health careworker).

The peptides and polypeptides of this invention can be administered to acell of a subject either in vivo or ex vivo. For administration to acell of the subject in vivo, as well as for administration to thesubject, the peptides and polypeptides of this invention can beadministered orally, parenterally (e.g., intravenously), byintramuscular injection, by intraperitoneal injection, subcutaneousinjection, transdermally, extracorporeally, topically or the like. Also,the peptide or polypeptide of this invention may be pulsed ontodendritic cells which are isolated or grown from patient cells,according to methods well known in the art, or onto bulk PBMC or variouscell subfractions thereof from a patient.

The exact amount of the peptide or polypeptide required will vary fromsubject to subject, depending on the species, age, weight and generalcondition of the subject, the particular peptide or polypeptide used,its mode of administration and the like. Thus, it is not possible tospecify an exact amount for every peptide or polypeptide. However, anappropriate amount can be determined by one of ordinary skill in the artusing only routine experimentation given the teachings herein (84).

As an example, to a subject diagnosed with HCV infection or known to beat risk of being infected by HCV, between about 50–1000 nM and morepreferably, between about 100–500 nM of a peptide and/or polypeptide ofthis invention can be administered subcutaneously and can be in anadjuvant, at one to three week intervals for approximately 12 weeks oruntil an evaluation of the subject's clinical parameters (symptoms,liver enzyme levels, HCV RNA levels) indicate that the subject is notinfected by HCV. Alternatively, a peptide and/or polypeptide of thisinvention can be pulsed onto dendritic cells at a concentration ofbetween about 10–100 μM and the dendritic cells can be administered tothe subject intravenously at the same time intervals. The treatment canbe continued or resumed if the subject's clinical parameters indicatethat HCV infection is present and can be maintained until the infectionis no longer detected by these parameters.

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the subject=s body according to standard protocolswell known in the art. The peptides or polypeptides of this inventioncan be introduced into the cells via known mechanisms for uptake ofpeptides and polypeptides into cells (e.g., phagocytosis, pulsing ontoclass I MHC-expressing cells, liposomes, etc.). The cells can then beinfused (e.g., in a pharmaceutically acceptable carrier) or transplantedback into the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject.

The nucleic acids and vectors of this invention can also be administeredto a cell of the subject either in vivo or ex vivo. The cell can be anycell which can take up and express exogenous nucleic acid and producethe peptides and polypeptides of this invention. Thus, the peptides andpolypeptides of this invention can be produced by a cell which secretesthem, whereby the peptide or polypeptide is produced and secreted andthen taken up and subsequently processed by an antigen presenting cellor other class I MHC-expressing cell and presented to the immune systemfor induction of an immune response. Alternatively, the peptides andpolypeptides of this invention can be directly produced in an antigenpresenting cell or other class I MHC-expressing cell in which thepeptide or polypeptide can be produced and processed directly andpresented to the immune system on the cell surface.

The nucleic acids and vectors of this invention can be administeredorally, parenterally (e.g., intravenously), by intramuscular injection,by intraperitoneal injection, transdermally, extracorporeally, topicallyor the like. In the methods described herein which include theadministration and uptake of exogenous DNA into the cells of a subject(i.e., gene transduction or transfection), the nucleic acids of thepresent invention can be in the form of naked DNA or the nucleic acidscan be in a vector for delivering the nucleic acids to the cells forexpression of the peptides or polypeptides of this invention. The vectorcan be a commercially available preparation or can be constructed in thelaboratory according to methods well known in the art.

Delivery of the nucleic acid or vector to cells can be via a variety ofmechanisms. As one example, delivery can be via a liposome, usingcommercially available liposome preparations such as LIPOFECTIN,LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen,Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison,Wis.), as well as other liposomes developed according to proceduresstandard in the art. In addition, the nucleic acid or vector of thisinvention can be delivered in vivo by electroporation, the technologyfor which is available from Genetronics, Inc. (San Diego, Calif.) aswell as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp.,Tucson, Ariz.).

As one example, vector delivery can be via a viral system, such as aretroviral vector system which can package a recombinant retroviralgenome (see e.g.,70,71). The recombinant retrovirus can then be used toinfect and thereby deliver to the infected cells nucleic acid encodingthe peptide or polypeptide. The exact method of introducing theexogenous nucleic acid into mammalian cells is, of course, not limitedto the use of retroviral vectors. Other techniques are widely availablefor this procedure including the use of adenoviral vectors (72),adeno-associated viral (AAV) vectors (73), lentiviral vectors (74),pseudotyped retroviral vectors (75) and vaccinia viral vectors (85), aswell as any other viral vectors now known or developed in the future.Physical transduction techniques can also be used, such as liposomedelivery and receptor-mediated and other endocytosis mechanisms (see,for example, 76). This invention can be used in conjunction with any ofthese or other commonly used gene transfer methods.

As one example, if the nucleic acid of this invention is delivered tothe cells of a subject in an adenovirus vector, the dosage foradministration of adenovirus to humans can range from about 10 to plaqueforming units (pfa) per injection, but can be as high as 10¹² pfu perinjection (77,78). Ideally, a subject will receive a single injection.If additional injections are necessary, they can be repeated at sixmonth intervals for an indefinite period and/or until the efficacy ofthe treatment has been established. As set forth herein, the efficacy oftreatment can be determined by evaluating the clinical parametersdescribed herein.

The exact amount of the nucleic acid or vector required will vary fromsubject to subject, depending on the species, age, weight and generalcondition of the subject, the particular nucleic acid or vector used,its mode of administration and the like. Thus, it is not possible tospecify an exact amount for every nucleic acid or vector. However, anappropriate amount can be determined by one of ordinary skill in the artusing only routine experimentation given the teachings herein (84).

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The nucleic acids and vectors of this invention can beintroduced into the cells via any gene transfer mechanism, such as, forexample, virus-mediated gene delivery, calcium phosphate mediated genedelivery, electroporation, microinjection or proteoliposomes. Thetransduced cells can then be infused (e.g., in a pharmaceuticallyacceptable carrier) or transplanted back into the subject per standardmethods for the cell or tissue type. Standard methods are known fortransplantation or infusion of various cells into a subject.

Parenteral administration of the peptides, polypeptides, nucleic acidsand/or vectors of the present invention, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. As used herein, “parenteral administration” includesintradermal, subcutaneous, intramuscular, intraperitoneal, intravenousand intratracheal routes. A more recently revised approach forparenteral administration involves use of a slow release or sustainedrelease system such that a constant dosage is maintained. See, e.g.,U.S. Pat. No. 3,610,795, which is incorporated by reference herein.

The efficacy of treating or preventing HCV infection by the methods ofthe present invention can be determined by detecting a clinicalimprovement of the subject=s symptoms, as would be well known to one ofskill in the art. Moreover, a decrease in the serum levels of liverenzymes such as SGOT and SGPT, as well as a decrease in viral RNA asdetermined by PCR assay can provide objective evidence of the efficacyof these methods.

The present invention additionally provides a method of activating acytotoxic T lymphocyte comprising contacting the lymphocyte with any ofthe peptides and/or polypeptides of this invention in the presence of aclass I MHC molecule. The class I MHC molecule can be produced insoluble form according to methods standard in the art or it can be onthe surface of a cell which expresses class I MHC. The conditions underwhich the CTL are contacted with the peptides and/or polypeptides in thepresence of the class I MHC molecule in order to activate the CTL arewell known in the art and are described in the Examples provided herein.Examples of cells which express class I MHC molecules include, but arenot limited to, dendritic cells, monocytes, macrophages, fibroblasts, Blymphocytes and T lymphocytes. Cytotoxic T lymphocyte cell lines can beobtained according to the methods set forth in the Examples providedherein. Activation of the CTL can be determined according to the methodsset forth in the Examples provided herein.

Thus, the present invention further provides a composition comprisingcytotoxic T lymphocytes activated by contact with any of the peptidesand/or polypeptides of this invention in the presence of a class I MHCmolecule. It would be understood that the composition can comprise apopulation of PBMC which includes activated CTL or the composition cancomprise a CTL line, as described in the Examples provided herein.

It is further contemplated that the compositions of the presentinvention can be used in diagnostic and therapeutic applications. Thus,the present invention provides a method of detecting the presence ofhepatitis C virus core polypeptide in a cell, comprising contacting thecell with the activated cytotoxic T lymphocytes of this invention underconditions whereby cytolysis of target cells can occur and detectingcytolysis of target cells, whereby the detection of cytolysis of targetcells indicates the presence of hepatitis C virus core polypeptide inthe cell. The cell can be any cell which can maintain infection by HCVand be recognized by the activated CTL of this invention.

A method of detecting the presence of hepatitis C virus core polypeptidein a cell is also provided, comprising contacting the cell with theactivated cytotoxic T lymphocytes of this invention under conditionswhereby cytokine production in the lymphocytes can occur and detectingcytokine production in the lymphocytes, whereby the detection ofcytokine production in the lymphocytes indicates the presence ofhepatitis C virus core polypeptide in the cell. The cell can be any cellwhich can maintain infection by HCV and be recognized by the activatedCTL of this invention.

Also provided herein is a method of detecting hepatitis C virus in asample, comprising: a) contacting the sample with a cell which issusceptible to infection by hepatitis C virus under conditions wherebythe cell can be infected by hepatitis C virus in the sample; b)contacting the cell of step (a) with the cytolytic T lymphocytes of thisinvention under conditions whereby cytolysis of target cells can occur;and c) detecting cytolysis of target cells, whereby the detection ofcytolysis of target cells indicates the presence of hepatitis C virus inthe sample.

The sample of this invention can be any sample in which infectious HCVcan be present. For example, the sample can be a sample removed from asubject, such as a body fluid, cells or tissue which can containinfectious HCV particles.

Furthermore, the present invention provides a method of detectinghepatitis C virus in a sample comprising: a) contacting the sample witha cell which is susceptible to infection by hepatitis C virus underconditions whereby the cell can be infected by hepatitis C Virus in thesample; b) contacting the cell of step (a) with the cytolytic Tlymphocytes of this invention under conditions whereby cytokineproduction can occur in the lymphocytes; and c) detecting cytokineproduction in the lymphocytes, whereby the detection of cytokineproduction in the lymphocytes indicates the presence of hepatitis Cvirus in the sample.

Additionally, the present invention provides a method of diagnosinghepatitis C virus infection in a subject comprising contacting cytotoxicT lymphocytes of the subject with any of the peptides or polypeptides ofthis invention in the presence of a class I MHC molecule underconditions whereby cytolysis of target cells can occur and detectingcytolysis of target cells, whereby the detection of cytolysis of targetcells indicates a diagnosis of hepatitis C virus infection in thesubject.

A method of diagnosing hepatitis C virus infection in a subject isfurther provided, comprising contacting cytotoxic T lymphocytes of thesubject with any of the peptides or polypeptides of this invention underconditions whereby cytokine production in the CTL can occur anddetecting cytokine production in the CTL, whereby the detection ofcytokine production in the CTL indicates a diagnosis of hepatitis Cvirus infection in the subject.

Cytotoxic T lymphocytes can be obtained from the subject as described inthe Examples set forth herein. The subject of this invention can be anyanimal which is susceptible to infection by HCV. For example, thesubject can be a chimpanzee and in a preferred embodiment, the subjectis a human.

The present invention also provides a method of determining a viral loadof hepatitis C virus in a subject comprising: a) serially diluting abiological sample from the subject which contains hepatitis C virus; b)contacting each serially diluted sample with a cell which is susceptibleto infection by hepatitis C virus under conditions whereby the cell canbe infected by hepatitis C virus in the sample; c) contacting the cellof step (b) with the cytolytic T lymphocytes of this invention underconditions whereby cytokine production can occur in the CTL; d)measuring the amount of cytokine production in the CTL; e) comparing theamount of cytokine production in the CTL of step (c) with the amount ofcytokine production in activated CTL contacted with cells infected withserially diluted control samples containing a known amount of hepatitisC virus; and f) determining the viral load of hepatitis C virus in thesubject from the comparison of step (e).

An additional method is provided, of determining a viral load ofhepatitis C virus in a subject comprising: a) serially diluting abiological sample from the subject which contains hepatitis C virus; b)contacting each serially diluted sample with a cell which is susceptibleto infection by hepatitis C virus under conditions whereby the cell canbe infected by hepatitis C virus in the sample; c) contacting the cellof step (b) with the cytolytic T lymphocytes of this invention underconditions whereby cytolysis of target cells can occur; d) measuring theamount of cytolysis of target cells; e) comparing the amount ofcytolysis of target cells of step (d) with the amount of cytolysis oftarget cells by activated cytotoxic T lymphocytes contacted with cellsinfected with serially diluted control samples containing a known amountof hepatitis C virus; and f) determining the viral load of hepatitis Cvirus in the subject from the comparison of step (e).

It is also contemplated that the viral load of HCV in a subject can bedetermined by a) contacting HCV-infected cells (e.g., liver cells, PBMC)with the cytolytic T lymphocytes of this invention; b) measuring theamount of cytolysis of target cells or cytokine production in the CTL;c) comparing the amount of cytolysis of target cells or cytokineproduction in the CTL of step (b) with the amount of cytolysis of targetcells or cytokine production in activated CTL contacted with cellsinfected with serially diluted control samples containing a known amountof hepatitis C virus; and d) determining the viral load of hepatitis Cvirus in the subject from the comparison of step (c).

The sample from the subject used in the methods of this invention can beany sample which can contain infectious HCV, including, but not limitedto, serum, plasma, liver tissue and PBMC.

On the basis of the information provided by the above methods, acalculation of HCV viral load in a subject can be carried out accordingto methods standard in the art for determining viral load (88).

Finally, the present invention also provides a method of determining theprognosis of a subject diagnosed with hepatitis C virus infection,comprising determining a viral load for the subject according to themethods of this invention, whereby a high viral load can indicate a poorprognosis and a low viral load can indicate a good prognosis.

A value of HCV viral load which would be a “high” viral load of HCV anda correlation with a high viral load with a subject=s prognosis can bedetermined according to methods well known in the art. For example, ahigh viral load as determined according to the methods of this inventioncan identify a subject who is in or likely to progress to an advancedstage of disease, as well as to provide a predictive indicator of asubject=s response to treatment, such as interferon therapy (88,89,90).

For the methods of this invention wherein cytolysis of target cells isdetected, detection of the cytolysis of target cells can be according toany method for detection of cytolysis of target cells now known or laterdeveloped. For example, the production of ⁵¹Cr-labeled target cells andan assay whereby cytolysis of target cells can be detected by release of⁵¹Cr release from a target cell, are described in the Examples providedherein. Other methods for detecting cytolysis can also be used in thesemethods, such as release of Europium or various dyes from appropriatelylabeled target cells, uptake of dyes into cells or detection ofapoptosis of target cells, according to methods standard in the art.

For the methods of this invention wherein the production of a cytokinein an activated cytolytic T lymphocyte is detected, the cytokine can be,but is not limited to, interleukin-2, interferon-γ,granulocyte/macrophage colony stimulating factor, interleukin-10,interleukin-4, interleukin-5 and/or interleukin-3. Detection of cytokineproduction can be by any method now known in the art, such as bycommercially available assay or by any other method later identified fordetecting the presence of a cytokine (86).

The present invention is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLES

Synthetic peptides. Peptides were prepared in an automated multiplepeptide synthesizer (Symphony; Protein Technologies, Inc.) using Fmocchemistry. Peptides were purified by reverse phase HPLC and the aminoacid sequence of each of the peptides was confirmed with an AppliedBiosystems 477A automated sequencer.

Cells. The T2 cell line is deficient in TAP1 and TAP2 transporterproteins and expresses low levels of HLA-A2.1 (50,51). The humanB-lymphoblastoid cell line HMYC1R was transfected with HLA-A2.1(C1R.A2.1). Cell line C1R.AAD (HMYC1R transfected with the HLA chimericmolecule containing α1 and α2 domains from human HLA-A2.1 and α3 frommouse H-2D^(d)) has been previously described (48). Cell lines weremaintained in complete T-cell medium (CTM; 1:1 mixture of RPMI:EHAA(Life Technologies; Grand Island, N.Y.) containing 10% fetal bovineserum (FBS), 4 mM glutamine, 100 U/ml penicillin, 100 Φg/ml streptomycinand 50 ΦM 2-mercaptoethanol).

Human subjects. Peripheral blood mononuclear cells (PBMC) were isolatedfrom a patient with chronic HCV infection. The patient had antiHCV-specific antibodies as detected with a commercially available assay(Ortho Diagnostics, Raritan, N.J.) and was HCV RNA positive by PCR.

Mice. Transgenic A2 Kb mice (Scripps Research Inst.) and transgenic AADmice (University of Virginia) were bred in the colony at BioCon Inc.(Rockville, Md.). These animals have been previously described (48,52)and they express α1 and α2 domains from human HLA-A2.1 molecule and α3domain from mouse H-2K^(b) and H-2D^(d) molecules, respectively.

Binding Assays. Peptide binding to HLA molecules was measured using theT2 mutant cell line according to a protocol previously described (53).T2 cells (3×10⁵/well) were incubated overnight in 96-well plates withculture medium (1:1 mixture of RPMI 1640: EHAA containing 2.5% FBS, 100U/ml penicillin, 100 Φg/ml streptomycin) with 10 Φg/ml β2-microglobulin(Sigma Chemical Co; St. Louis, Mo.) and different peptideconcentrations. The next day, cells were washed twice with cold PBScontaining 2% FBS and incubated for 30 min at 4 EC with anti HLA-A2.1BB7.2 monoclonal antibody (1/80 dilution from hybridoma supernatant) and5 Φg/ml FITC-labeled goat anti mouse Ig (Pharmingen; San Diego, Calif.).Cells were washed twice after each incubation and HLA-A2.1 expressionwas measured by flow cytometry on a FACScan (Becton Dickinson). HLA-A2.1expression was quantified as fluorescence index (FI) according to theformula: FI=(mean fluorescence with peptide−mean fluorescence withoutpeptide)/mean fluorescence without peptide. Background fluorescencewithout BB7.2 antibody was subtracted for each individual value. Inorder to compare the different peptides, FI_(0.5), the peptideconcentration that increases HLA-A2.1 expression by 50% over no peptidecontrol background, was calculated from the titration curve for eachpeptide.

Generation of human CTL. Cytotoxic T lymphocytes (CTL) lines weregenerated according to the protocol previously described (12,14). PBMCfrom a patient chronically infected with HCV were separated byFicoll-Hypaque and 10⁶ cells were stimulated in 400 Φl CTM in 48-wellplates with 2×10⁶ autologous cells pulsed previously for 3 h with 10 ΦMpeptide in CTM. After 3 days, the same volume of CTM with IL-2 (10%vol./vol.) was added to each well and the cells were further expanded onday six with CTM containing IL-2. This cycle was repeated every tendays. For the first three cycles, 2×10⁶ peptide-pulsed, irradiated (3000rads) autologous PBMC were used as antigen presenting cells (APC) and insubsequent cycles, CTL were stimulated with 3×10⁵ peptide-pulsed,irradiated (3000 rads) autologous Con A blasts and 1.5×10⁶ irradiatedallogeneic PBMC. Con A blasts were obtained by stimulating PBMC with 10Φg/ml Concanavalin A and expanding with CTM containing 50 U/ml IL-2 and1 U/ml IL-6. Con A blasts were stimulated with Con A every 8–10 days.The stimulation of PBMC with peptide results in the expansion of peptideantigen-specific CTL, while non-specific cells that are not stimulateddie out, resulting in a CTL line that is specific for the peptide. Thisenrichment, resulting in an essentially homogenous population of peptideantigen-specific CTL occurs after about 3–5 cycles of stimulation. Usingthis procedure, a CTL line, ViT2, was raised from a patient withchronic, active HCV infection.

Generation of mouse CTL. Eight to twelve week old mice were immunizedsubcutaneously (s.c.) in the base of the tail with 100 Φl of an emulsioncontaining 1:1 incomplete Freund's adjuvant (IFA) and PBS solution withpeptides (antigens) and cytokines (50 nmol CTL epitope, 50 nmol HBV core(aa) 128–140 helper epitope, 3 Φg IL-12 and 3 Φg GM-CSF) (54). Mice wereboosted 2 weeks later and spleens removed 10–14 days after the boost.Immune spleen cells (2.5×10⁶/well) were stimulated in 24-well plateswith autologous spleen cells (5×10⁶/well) pulsed for 2 h with 10 ΦM CTLepitope peptide in CTM with 10% T-STIM (Collaborative BiomedicalProducts, Bedford, Mass.). After further in vitro stimulation withpeptide-pulsed, syngeneic spleen cells, CTL lines were produced andmaintained by weekly restimulation of 10⁶ CTL/well with 2×10⁵peptide-pulsed, irradiated (10,000 rads) C1R.AAD cells and 4×10⁶ C57/B16irradiated (3000 rads) spleen cells as feeders.

Cytotoxicity assay. CTL activity was measured using a 4 h assay with⁵¹Cr-labeled target cells. Target cells (10⁶) were pulsed in 100 Φl CTMwith or without 10 ΦM peptide and 150 ΦCi ⁵¹Cr for 2 h, washed threetimes and added to the plates containing different amounts of CTL(effector) cells in a final volume of 200 Φl CTM. In peptide titrationassays, target cells were pulsed with ⁵¹Cr for 2 h, washed three timesand added to the plates with different peptide concentrations. Effectorcells were added 2 h later and the supernatants were harvested andcounted after an additional 4 h incubation. The percentage of specific⁵¹Cr release was calculated as: 100×(experimental release−spontaneousrelease)/(maximum release−spontaneous release). Spontaneous release wasdetermined from target cells incubated without effector cells andmaximum release was determined in the presence of 5% Triton X-100.C1R.A2.1 and C1R.AAD lines and AAD and A2 Kb Con A blasts were used astarget cells. Con A blasts were prepared by culturing 3×10⁶ spleen cellsin 2 ml of CTM in the presence of 2 Φg/ml of Con A in 24-well cultureplates. After 2 days, cells were harvested and processed for labeling asdescribed.

C7A2 Ala-substituted peptides binding to HLA-A2.1 molecules. The bindingaffinity of wild-type C7A2 peptide (C7A2-WT) was evaluated with a T2binding assay (53), which measures the cell surface stabilization ofHLA-A2.1 molecules after incubation with peptide. In order to comparethe different peptides, a fluorescence index value of 0.5 (FI_(0.5)),the peptide concentration that increases HLA-A2.1 expression by 50%, waschosen as a way to compare titrations of the peptides and relativeaffinity for MHC molecules. Using this method, F_(0.5) of 10 ΦM wascalculated for C7A2-WT.

In a first set of experiments to define key functional residues,peptides with alanine substitutions at each one of the positions weresynthesized and tested in binding assays (Table I). C7A2-WT has thetypical motif for binding to HLA-A2.1, with L and V at positions 2 and 9respectively (22,23). Binding experiments with these alanine-substitutedpeptides showed (FIG. 1A) that peptide 2A had lost binding ability, inaccordance with the anchor character of this position, and also 9A hadimpaired binding, although not as much as 2A, since Ala can function asa weak anchor at position 9. Other substitutions that decreased bindingwere 6A and 7A, indicating the importance of these residues as secondaryanchor positions. Peptides 4A and 8A had higher affinity, with FI_(0.5)around 2 ΦM. Substitutions at positions 1, 3 and 5 had no effect onpeptide binding.

Binding of peptides with substitutions at position 1. C7A2-WT hasaspartic acid, a negatively charged residue, in position 1 (28).Substitution by Ala at this position, although replacing the chargedresidue, did not improve binding, so the original D was replaced byother residues. Peptides with aromatic amino acids at this position (Fand H), as well as N (which eliminates the negative charge but keeps thesize), or S and T, small polar amino acids which can form hydrogenbonds, were synthesized.

Peptides 1N, 1H and 1T had improved binding affinity, whereas 1F had aFI_(0.5) higher than C7A2-WT, indicating lower affinity. Solubilityproblems with IF may account for impaired binding ability in this case.

Binding of peptides with substitutions at other positions. Substitutionby A at position 7 yielded a peptide with impaired binding ability. Thenatural amino acid at this position is P, which is known to induce turnsin peptide chains. In order to determine whether the A replacementchanged the structure of the peptide chain or eliminated the side chainresponsible for interaction with HLA-A2.1, 7G was synthesized, therebyintroducing an amino acid that also contributes to β turns as does P,but lacks the side chain. 7V was also synthesized, with an aliphaticside chain bulkier than A. None of these peptides bound to HLA-A2.1(FIG. 1B), demonstrating a specific role of the P side chain in thebinding to HLA-A2.1.

Position 3 was replaced by W and by K, L, I and V, which are charged andaliphatic residues. Binding was improved with 3W (FIG. 1B), but it wascompletely abolished in 3K. Peptide 3V had impaired binding, whereas 3Land 31 were similar to C7A2-WT.

Replacement of position 8L with a V introduced an aliphatic residuesimilar to A and L (amino acid present in C7A2-WT), but with anintermediate size between them. Peptide 8V is one of the few sequencevariations that can be found within this epitope and is common in viralisolates belonging to HCV genotypes 2a and 2b (56,57). Peptide 8V had anFI_(0.5) of 15 ΦM, which was in the range of C7A2-WT.

Recognition of C7A2-variant peptides by human CTL. To identify residuesinvolved in CTL recognition, a CTL line specific for C7A2 wasestablished as described herein from PBMC from an HCV infected patientafter several rounds of C7A2-WT peptide stimulation. Lytic activity ofthese CTL was tested with target cells incubated with differentconcentrations of each peptides to study the recognition of thedifferent peptide variants and titrate T-cell avidity. TheAla-substituted peptides, 2A and 9A were not recognized (FIG. 2A), inaccordance with the anchor character of these positions. Also, 3A wasnot recognized, despite good HLA-A2.1 binding, indicating the epitopiccharacter of this position. Peptides 4A, 5A, 6A and 7A, althoughrecognized at the highest concentrations, showed poorer recognition thanC7A2-WT, as a consequence in some cases, of poorer MHC binding (6A and7A), or otherwise of impaired T-cell recognition (4A and 5A). Peptides1A and 8A showed higher levels of lysis than C7A2-WT and titrated at10-fold lower concentrations.

Of the non-Ala substituted peptides (FIG. 2B), none was recognized bythe CTL at the concentrations tested (including those having highbinding affinity, such as IN or IT), with the exception of 8V, whichbehaved similarly to C7A2-WT, and 1H, that induced marginal lysis at thehighest concentration tested.

Recognition of C7A2-variant peptides by HLA-A2.1 transgenic mice CTL.AAD transgenic mice express α1 and α2 domains from the human HLA-A2.1molecule and α3 domain from the mouse D^(d) molecule (48). Transgenicmice expressing human HLA-A2.1 molecules have been described as a modelof presentation and recognition of several HLA-A2.1-restricted antigens(9,37,47,58–60) and allow testing of immunogenicity in the context of ahuman HLA molecule prior to immunizing humans. In order to use thepeptide antigens in this model, recognition of the different C7A2peptide variants by murine CTL in vitro was studied prior to testing theimmunogenicity of the different peptides in vivo.

CTL lines AAD.10 and AAD.1 were induced by immunization with peptideC7A2-WT together with a helper epitope presented by the H-2^(b) class IIMHC molecules of this mouse strain and GM-CSF and IL-12 according to themethod of Ahlers et al. (54) and as described herein. After ten totwelve in vitro stimulations with C7A2-WT, these cell lines were testedagainst the whole panel of C7A2-substituted peptides in a lytic assay tocompare the recognition of the different peptides with that by the humanCTL line.

Of the Ala-substituted peptides (FIGS. 3A and 3C), 1A and 8A were ableto sensitize target cells in a concentration range around that ofC7A2-WT, 1A being more effective than 8A for both lines. The otherpeptides titrated at several 10-fold concentrations higher. Peptides 5A,7A and 2A required the highest concentrations to induce significantlysis, whereas 3A, 6A, 4A and 9A titrated at intermediateconcentrations. Position 5 was clearly shown to be an epitopic residueand almost no significant lysis was obtained with target cellssensitized with peptide 5A, as it had been observed for position 3 inthe human CTL line. Peptide 3A titrated at a lower concentration,suggesting that in the case of the murine HLA-A2.1-restricted CTL lines,position 3 is not involved in TCR binding as clearly as in the humanline. Finally, peptides with substitutions at positions 4 and 6 wererecognized as in the case of the human CTL line.

Peptides with substitutions by other amino acids and positive binding toHLA-A2.1 were also tested for recognition by AAD mouse CTL lines (FIGS.3B and 3D). These peptides contained mainly substitutions at position 1,except for peptides 8V and 3W. Surprisingly, with the exception of IS,that required a high peptide concentration to sensitize target cells,all the peptides with substitutions at position 1 were recognized by AADmouse CTL lines in a concentration range similar to that observed forC7A2-WT and some peptides, such as 1F, induced a higher response withline AAD.10. Peptide 8V was recognized at higher concentrations thandetermined for C7A2-WT, whereas peptide 3W was not recognized at any ofthe concentrations tested.

In vivo immunogenicity of C7A2-derived peptides in HLA-A2.1 transgenicmice. After studying the recognition of C7A2-derived peptides by humanand AAD transgenic murine CTL, the in vivo immunogenicity of thesepeptides was studied in the AAD transgenic mouse model. Different groupsof animals were immunized with the substituted CTL peptides (epitopes)in conjunction with a helper epitope and cytokines as described aboveand the ability to induce an immune response was tested in CTLcytotoxicity assays. Included in these assays were some of the peptideswith positive binding that were recognized by AAD mouse CTL lines. Asshown in FIG. 4, peptides C7A2-WT and 8A were able to induce clear CTLresponses, whereas 1A could induce only a marginal response. Incontrast, peptides 1N and 1F were unable to induce any measurable CTLresponse.

Induction of CTL immune response against C7A2-WT by C7A2-WT and 8Apeptides in HLA-A2.1 transgenic mice. Among all the peptides tested inbinding assays to HLA-A2.1 and for recognition by human and transgenicmice CTL, peptide 8A was the most promising. This peptide was able tobind at lower concentrations than C7A2-WT, sensitized human and mousetarget cells in the same range or even at lower concentrations thanC7A2-WT and in immunization experiments, induced higher levels of lysis.Therefore, the ability of 8A to induce a CTL immune response againstC7A2-WT was tested in the next set of experiments.

Each peptide was used to immunize two groups of animals, with 50 or 15nmol CTL epitope peptide as described herein. After two immunizations,spleen cells were removed and stimulated in vitro with the same peptideused for immunization and lytic activity against C7A2-WT (and 8A in thegroups immunized with 8A) was tested. As shown in FIG. 5, two of thefour animals immunized with C7A2-WT were able to respond to thispeptide. In the animals immunized with 8A, strong responses to C7A2-WTcould be detected, together with positive responses to 8A itself, evenin the group of mice immunized with 15 nmol peptide (FIG. 5B).

In a second group of experiments, A2 Kb transgenic mice, that express achimeric HLA molecule with α1 and α2 domains from human HLA-A2.1 and α3domain from mouse K^(b), were immunized with 50 nmol C7A2-WT or 8Apeptide and the ability of these peptides to induce a CTL immuneresponse recognizing C7A2-WT was tested. FIG. 6 shows that, as seen withthe AAD mice, C7A2-WT induced a low response, whereas the immunizationwith 8A induced a higher level of response by CTL that recognized bothC7A2-WT and 8A, even with a lower E/T ratio in the CTL assay (70:1 vs.120:1).

CTL avidity for a target peptide-MHC complex, defined as the sensitivityto detect low densities of peptide-MHC complex on the surface of targetor stimulator cells, has been shown to make a substantial difference inthe ability of CTL to clear a virus infection in vivo, in two murinemodel systems (61,62). Therefore, to determine whether the CTL raisedagainst the modified peptide 8A were of as high avidity against targetspresenting the wild-type peptide as would be CTL raised against thewild-type peptide itself, C7A2-WT was titrated on target cells and the(lysis) killing by short term CTL lines (which received 34 cycles of invitro stimulation with peptide) raised against this peptide or against8A was compared (FIG. 7). The CTL raised against 8A killed targetsexpressing the C7A2-WT at concentrations more than 100-fold lower thanwere required to get comparable levels of killing by the CTL raisedagainst the C7A2-WT itself. Thus, use of the modified peptide in thiscase increases not only immunogenicity, but also the avidity of the CTLthat are produced, an additional advantage for the potential efficacy ofa vaccine.

Recognition of peptide 8Vby CTL from HLA-A2.1 transgenic mice immunizedwith 8A. As mentioned above, one of the peptides recognized by bothhuman and mouse C7A2-WT specific CTL lines is peptide 8V. This peptideis common in viral isolates from HCV genotypes 2a and 2b (56,57). Since8A has a change in the same position, it was an interesting point toknow if CTL induced by immunization with 8A were cross reactive not onlywith C7A2-WT, which corresponds to viral sequences from genotypes 1a, 1band 3a, but also with peptide 8V, belonging to viral sequences fromgenotype 2. Results in FIGS. 8A and 8B show that CTL induced byimmunization with 8A recognize peptide 8V both in AAD mice (FIG. 8A) andin A2 Kb mice (FIG. 8B) in a manner similar to the recognition ofC7A2-WT.

Enhanced epitope C7A2-8A in DNA vaccine against HCV. To produce a DNAvaccine encoding the enhanced epitope of this invention, a DNA plasmidvaccine expressing HCV core protein was first prepared. The vector AC7,encoding the entire nucleocapsid (core) region of HCV H (1a) strain (91)(amino acids 1–191) was constructed. The sequence was amplified bypolymerase chain reaction (PCR) and inserted into the Hind III and BamHI sites of the eukaryotic expression vector pWRG7020 under thetranscriptional control of the cytomegalovirus early promoter.(Agracetus Inc., Middleton, Wis.). Direct sequencing of the insert DNAusing the fluorescent dye terminator cycle method on an ABI 310automated sequencer (Applied Biosystems, Forester City, Calif.) showedthe expected sequence in frame. Plasmid AC7 was amplified in Escherichiacoli, purified using Quiagen purification kit (Quiagen Inc., Chatsworth,Calif.), and maintained at −20° C. until use.

Transient expression of HCV core protein in cell culture was confirmedin human 293 cells, as previously described (92). Briefly, the plasmidwas transfected into 293 cells using lipofectamine (GIBCO/BRL) andprotein expression was analyzed by indirect immunofluorescence stainingand immunoblotting using a specific mouse monoclonal antibodyrecognizing HCV core (6G7; Stanford University, Palo Alto Va. MedicalCenter, Palo Alto, Calif.; ref 93). For immunization, AC7 wasprecipitated with 3M sodium acetate and ethanol and redissolved inphosphate buffered saline (PBS).

A variant of this plasmid was then constructed with the substitution ofan Ala codon for a Leu codon at amino acid residue position 8 of theepitope C7A2. The codon at nucleotides 756–758 in the core of the HCVgenome region was changed from CTC (Leu) to GCG (Ala) by PCRamplification. Forward (8AF: TCATGGGGTACATACCGGCGGTC nt. 739–761) (SEQID NO:5) and reverse (8AR: TCCAAGAGGGGCGCCGACCGCCG nt. 776–754) (SEQ IDNO:6) primers were synthesized containing the 8A sequence at thislocation. Two overlapping PCR products were generated using primers 243F(AAGACTGCTAGCCGAGTAGTG nt. 243–263) (SEQ ID NO:7) and 8AR or 8AF and980R (CACAATACTCGAGTTAGGGC nt. 980–961) (SEQ ID NO:8). Following gelisolation of the PCR products, an assembly PCR was carried out using 100ng of each of the fragments and primers to generate the full length coresequence with Hind III and Not I restriction sites engineered at the 5′and 3′ ends, respectively. This product was cloned into the vectorWRG7020 and the sequence verified by the fluorescent dye terminatorcycle method as described above.

AAD mice, transgenic for the human HLA molecule HLA-A2.1 with thealpha-3 domain from H-2D^(d) were immunized i.m. in the quadriceps majoron days 0, 14, and 28 with 100, 30, or 10 μg doses of AC7 or AC7–8A, or100 μg pWRG7020 (plasmid without the HCV core gene insert, as controlmock vaccine) dissolved in PBS solution. Four weeks after the lastimmunization, spleen cells from AC7-immunized mice were stimulated invitro with C7A2 peptide and those from AC7–8A-immunized mice werestimulated in vitro with C7A2-8A peptide, and all were assayed for CTLactivity against C1R-AAD targets coated with the C7A2 peptide. TheCIR-AAD cells express only the chimeric HLA-A2.1 class I molecule and noclass II MHC molecules, so the CTL activity was restricted to class IHLA-A2.1. As shown in FIG. 9, at the 30 μg dose, all three miceimmunized with the AC7–8A DNA vaccine had substantially higher CTLactivity than the three mice immunized with the unmodified AC7 DNAvaccine. The same effect was seen at the 100 and 10 μg doses, but themagnitude of the difference was less.

To test efficacy for clearing a viral infection, AAD mice, transgenicfor the human HLA-A2.1 molecule with the alpha-3 domain of H-2D^(d) tobe compatible with murine CD8, were infected with a recombinant vacciniavirus expressing the entire HCV core protein. This core protein gene didnot contain the 8A modification, but rather was representative of thewild-type HCV. The vaccinia virus preferentially replicates in theovaries and the titer of vaccinia in the ovaries (pfu/ovary) was used asa measure of the ability of the vaccine to reduce the level of viralinfection. Mice were immunized on days 0, 14, and 28, as above, and twoweeks after the last immunization, the mice were challengedintraperitoneally with 1×10⁷ pfu of rVV-core (recombinant vaccinia virusexpressing HCV core). Mice were sacrificed for harvest of ovaries 5 daysafter challenge. Some mice were treated 7, 6, 5, and 4 days beforesacrifice with 0.5 mg anti-CD8 antibody intraperitoneally in order todetermine the dependence of protection on CD8⁺ cells. The ovaries (wherethis vaccinia virus preferentially replicates; 61,95) were harvested,homogenized, sonicated, and assayed for rVV-core titer by plating10-fold dilutions on BSC-1 indicator cells, and staining with 0.075 w/v% crystal violet. The minimal detectable level of virus was 100pfu/ovary. Three mice were used in each group.

As shown in Table I, at the optimal 30 μg dose of vaccine, AC7unmodified core vaccine reduced the titers not quite 5 logs, compared tothe control mice immunized with mock vaccine (mice #1–3, with titers of3–4×10³, compared to mice #13–15, with titers of 1.6–2.8×10⁸). However,mice #7–9, immunized with the modified DNA vaccine AC7–8A, had titersreduced to undetectable levels. With both DNA vaccines, treatment of themice with anti-CD8 antibody completely abrogated protection (mice #4–6and #10–12), indicating that the protection was completely dependent onCD8⁺ cells (CTL). Thus, the modified HCV-core DNA vaccine with the8A-modified epitope is more effective than the wild-type HCV-core DNAvaccine in inducing CD8⁺ CTL restricted to the human HLA-A2 molecule andin inducing CD8⁺ protection against a surrogate virus expressing the HCVcore protein. Although the present process has been described withreference to specific details of certain embodiments thereof, it is notintended that such details should be regarded as limitations upon thescope of the invention except as and to the extent that they areincluded in the accompanying claims.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

TABLE I Mouse DNA Vaccine Dose (Φg) Anti-CD8 pfu/ovary 1 AC7 30 −   3 ×10³ 2 AC7   4 × 10³ 3 AC7   3 × 10³ 4 AC7 30 + 2.5 × 10⁸ 5 AC7 3.9 × 10⁸6 AC7 1.5 × 10⁸ 7 AC7-8A 30 − <100 8 AC7-8A <100 9 AC7-8A <100 10 AC7-8A30 + 1.8 × 10⁸ 11 AC7-8A 1.5 × 10⁸ 12 AC7-8A 1.3 × 10⁸ 13 Mock 100  −2.4 × 10⁸ 14 Mock 2.8 × 10⁸ 15 Mock 1.6 × 10⁸

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1. A composition comprising an isolated peptide having the amino acidsequence of SEQ ID NO: 1 and a pharmaceutically acceptable carrier.
 2. Acomposition comprising an isolated hepatitis C virus core polypeptidecomprising an L→A substitution at amino acid position 139 and apharmaceutically acceptable carrier.
 3. A composition comprising anisolated hepatitis C virus core polypeptide having the amino acidsequence of SEQ ID NO:2 and a pharmaceutically acceptable carrier.
 4. Acomposition comprising an isolated fragment of a hepatitis C virus corepolypeptide having fewer amino acids than the entire hepatitis C viruscore polypeptide, comprising the amino acid sequence of SEQ ID NO: 1 anda pharmaceutically acceptable carrier.
 5. A composition comprising anisolated nucleic acid encoding the peptide of claim 1 and apharmaceutically acceptable carrier.
 6. A composition comprising anisolated nucleic acid encoding the peptide of claim 2 and apharmaceutically acceptable carrier.
 7. A composition comprising anisolated nucleic acid encoding the peptide of claim 3 and apharmaceutically acceptable carrier.
 8. A composition comprising anisolated nucleic acid encoding the peptide of claim 4 and apharmaceutically acceptable carrier.
 9. A composition comprising anisolated vector comprising the nucleic acid of claim 5 and apharmaceutically acceptable carrier.
 10. A composition comprising anisolated vector comprising the nucleic acid of claim 6 and apharmaceutically acceptable carrier.
 11. A composition comprising anisolated vector comprising the nucleic acid of claim 7 and apharmaceutically acceptable carrier.
 12. A composition comprising anisolated vector comprising the nucleic acid of claim 8 and apharmaceutically acceptable carrier.